The long-term goal of this work is to understand the molecular basis of cellular morphogenesis. Oriented cell division and the establishment of cell polarity are essential features in the development of many organisms. Cells of the budding yeast Saccharomyces cerevisiae also exhibit simple, defined patterns of oriented cell divisions by choosing a specific bud site depending on their cell type. Previous genetic analyses have resulted in identification of several genes involved in yeast cell morphogenesis. Proteins encoded by these genes belong to highly conserved families of proteins in eukaryotes. These include the Ras-like small GTP binding protein Bud1, the GTPase Activating Protein (GAP) Bud2, and the Guanine nucleotide Exchange Factor (GEF) Bud5. These three proteins comprise a functional GTPase module that determines the site of budding by guiding another group of proteins that are necessary for bud formation. The second group of proteins includes the Rho-like GTPase Cdc42, Cdc24, and Bem1. It is believed that the spatial control of budding and actin polarization involves a cascade of GTPases, Bud1 controlling Cdc42, which ultimately controls the actin cytoskeleton. This proposal aims to determine the function of the Bud1 GTPase module in polarity establishment during yeast budding. Specific aims are to study: (i) how components of the Bud1 GTPase module interact with and modulate key regulators of the cytoskeleton to direct polarity establishment and (ii) how the Bud1 GTPase module recognizes the intrinsic spatial cues to establish the cell-type-specific budding pattern. To accomplish these goals, genetic, biochemical, and cell biological approaches will be undertaken. Recent work on Ras-related GTPases in yeast and mammalian cells have brought to our attention a new regulatory mechanism for cellular morphogenesis. Understanding the functions of the Bud1 GTPase cycle will help us to unravel the series of morphogenetic events culminating in polarized actin organization by a GTPase cascade. This work will also help us to understand the mechanism of genesis of human cancer since mutations in human homologs of these yeast proteins have been found in human tumor.